varieties with a high content of RebM, are disclosed Further provided are methods for producingplants having a high RebM content by negatively regulating certain genes selecting the resulting plants, and breeding with such plants to confer such desirable Reb M phenotypes to plant progeny.
Legal claims defining the scope of protection, as filed with the USPTO.
. Aplant comprising at least one disrupted negative regulator gene in the rebaudioside A to rebaudioside M conversion pathway, and wherein the negative regulator gene is selected from the group consisting of SEQ ID NO:9 and SEQ ID NO:10.
. A method of increasing the rebaudioside M content in aplant by inducing a disruption in at least one negative regulator gene function in the plant, wherein said negative regulator gene is selected from the group consisting of SEQ ID NO:9 and SEQ ID NO:10.
. A plant part of theplant of, wherein the plant part is selected from the group consisting of leaves, pollen, embryos, cotyledons, hypocotyl, meristematic cells, ovules, seeds, cells, roots, root tips, pistils, anthers, flowers, and stems.
. A plant, plant part, or cell produced by the method of, comprising in its genome or more genes affecting metabolism and selected from the group consisting of SEQ ID NO:9 and SEQ ID NO:10.
. A method of producing aplant comprising the steps of:
Complete technical specification and implementation details from the patent document.
This application is a divisional application which claims the benefit of priority to U.S. application Ser. No. 17/701,154, now U.S. Pat. No. 11,844,323 issue date of Dec. 19, 2023 which is a divisional of U.S. application Ser. No. 16/491,470, now U.S. Pat. No. 11,284,577 issued Mar. 29, 2022 which is a 371 National Stage entry of and claims the benefit of priority to PCT Application No. PCT/US2018/021389 filed on Mar. 7, 2018, which claims the benefit of priority to U.S. Provisional Application No. 62/468,937, filed on Mar. 8, 2017, the entire contents of which are incorporated herein by reference for all purposes.
The Sequence Listing associated with this application is filed in electronic format via PatentCenter under the xml file PURC08USG1 Sequence Listing and is hereby incorporated by reference into the specification in its entirety.
is an important and valuable field crop for the production of sweeteners, sugar substitutes, and other consumable ingredients. Thus, a continuing goal ofplant breeders is to develop stable, high yieldingcultivars ofspecies that are agronomically sound. The reasons for this goal are to maximize the amount and quality of the sweeteners, sugar substitutes, and other consumable ingredients. To accomplish this goal, thebreeder must select and develop plants that have the traits that result in superior cultivars. All references cited herein are incorporated by reference in their entirety.
The development of newcultivars requires the evaluation and selection of parents and the crossing of these parents. The lack of predictable success of a given cross requires that a breeder, in any given year, make several crosses with the same or different breeding objectives.
The foregoing examples of the related art and limitations related therewith are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to those of skill in the art upon a reading of the specification.
The following embodiments and aspects thereof are described and illustrated in conjunction with products and methods, which are meant to be exemplary and illustrative, not limiting in scope. In various embodiments, one or more of the above-described problems have been reduced or eliminated, while other embodiments are directed to other improvements.
It is believed that the conversion of rebaudioside A to rebaudioside M in the plant requires minimal steps to accomplish. However, knownspecies contain very little rebaudioside M compared to rebaudioside A, suggesting, that the conversion pathway is subject to negative effect of certain regulating elements in these plants. By identifying these negative regulators and disrupting their functionality, it is believed that the conversion pathway from rebaudioside A to rebaudioside M can occur more readily, resulting in a higher yield of rebaudioside M in theleaf
An embodiment of the present disclosure is directed to producingplant cultivars which produce high levels of rebaudioside M as compared to native or commercially knownplants such asBertoni andMorita.
Another embodiment discloses the disruption of negative regulators of rebaudioside M biosynthesis. By disrupting the function of these negative regulators inplants can be produced that are able to produce higher levels of rebaudioside M as compared to differentvarieties.
Another embodiment discloses aplant comprising at least one disrupted negative regulator gene in the rebaudioside A to rebaudioside M conversion pathway.
Another embodiment discloses a method of increasing the rebaudioside M content in aplant by inducing a disruption in at least one negative regulator gene function in the plant, wherein said negative regulator gene is selected from the group consisting of genes affecting metabolism, signal transduction and gene regulation, and other novel uncategorized genes.
Another embodiment discloses metabolism genes, wherein said genes are comprised of genes affecting sugar metabolism, mono-oxygenase content, terpene metabolism, aminotransferase metabolism, multi-antimicrobial extrusion protein metabolism, and methionine metabolism.
Another embodiment discloses metabolism genes, wherein said genes are selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10.
Another embodiment discloses a plant, or a plant part thereof, of theplant of the present application, consisting of leaves, pollen, embryos, cotyledons, hypocotyl, meristematic cells, ovules, seeds, cells, roots, root tips, pistils, anthers, flowers, and stems.
Another embodiment discloses signal transduction and gene regulation, wherein said genes are comprised of abiotic stress genes and biotic stress genes.
Another embodiment discloses signal transduction and gene regulation genes, wherein said genes are selected from the group consisting of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16.
Another embodiment discloses novel uncategorized genes, wherein said genes are selected from the group consisting of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20.
Another embodiment discloses a method for producing a high Rebaudioside Mplant comprising: (a) screening a population ofplants for a mutation in at least one of the following sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20; (b) selecting a firstplant having said at least one mutation; (c) crossing the first selectedplant having at least one mutation with a secondplant; (d) screening theoffspring for plants having high Rebaudioside M; and (e) selecting aplant having high Rebaudioside M.
SEQ ID NO: 1 discloses the endo-1,3; 1,4-beta-D-Glucanase gene (STKS #1).
SEQ ID NO: 2 discloses the glucose-6-phosphate dehydrogenase gene (STKS #2).
SEQ ID NO: 3 discloses the cytochrome P450 716B1-like gene (STKS #3).
SEQ ID NO: 4 discloses the putative flavonoid 3′-hydroxylase cytochrome P450 gene (STKS #4).
SEQ ID NO: 5 discloses the KAH-like cytochrome P450 gene (STKS #5).
SEQ ID NO: 6 discloses the Isoprenoid Biosynthesis Cl Superfamily gene (STKS #6).
SEQ ID NO: 7 discloses the vestitone reductase-like gene (STKS #7).
SEQ ID NO: 8 discloses the aminotransferase gene (STKS #8).
SEQ ID NO: 9 discloses the MATE efflux family protein gene (STKS #9).
SEQ ID NO: 10 discloses the methionine adenosyltransferase 2 subunit beta-like gene (STKS #10).
SEQ ID NO: 11 discloses the SAL1 phosphatase-like isoform gene (STKS #11).
SEQ ID NO: 12 discloses the nitrate-dependent transcription regulator gene (STKS #12).
SEQ ID NO: 13 discloses the LURP1-like domain-containing protein gene (STKS #13).
SEQ ID NO: 14 discloses the RGC2 Resistance Protein gene (STKS #14).
SEQ ID NO: 15 discloses the LRR Receptor Kinase gene (STKS #15).
SEQ ID NO: 16 discloses the Wall Associated Receptor Kinase gene (STKS #16).
SEQ ID NO: 17 discloses a DNA sequence of unknown function (2672) (STKS #17).
SEQ ID NO: 18 discloses a DNA sequence of unknown function (5237) (STKS #18).
SEQ ID NO: 19 discloses a DNA sequence of unknown function (10666) (STKS #19).
SEQ ID NO: 20 discloses a DNA sequence of unknown function (13209) (STKS #20).
In the description and tables, which follow, a number of terms are used. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided. All rebaudioside contents are represented as a percentage of the dry weight of leaves.
High rebaudioside A: As used herein, plants described as having high rebaudioside A have a rebaudioside A content of greater than or equal to 9%, a rebaudioside D content of less than or equal to 0.3%, and a rebaudioside M content of less than or equal to 0.2%.
High rebaudioside D: As used herein, plants described as having high rebaudioside D have a rebaudioside D content of greater than or equal to 0.6% and a rebaudioside D/total steviol glycoside greater than or equal to 8%.
High rebaudioside D and high rebaudioside M: As used herein, plants described as having rebaudioside D and high rebaudioside M content have a rebaudioside D content of greater than or equal to 0.6% and a rebaudioside D/total steviol glycoside greater than or equal to 8%, and a rebaudioside M content that is greater than or equal to 0.5%.
High rebaudioside M: As used herein, plants described as having high rebaudioside M have a rebaudioside M content that is greater than or equal to 0.5%.
High stevioside: As used herein, plants described as having high stevioside have a stevioside content of greater than or equal to 7%, a rebaudioside D content of less than or equal to 0.3%, and a rebaudioside M content of less than or equal to 0.2%.
High stevioside and high rebaudioside A: As used herein, plants described as having high stevioside and high rebaudioside A have a rebaudioside D/total steviol glycoside less than or equal to 7.60% and a rebaudioside M/total steviol glycoside less than or equal to 1.9%.
Marker: As used herein, a “marker” is an indicator for the presence of at least one polymorphism, thus a marker can be the nucleotide sequence itself, or a probe, for example.
Plant: As used herein, the term “plant” includes reference to an immature or mature whole plant, including a plant that has been processed for steviol glycosides. Seed or plant part that will produce the plant is also considered to be the plant.
Plant Part: As used herein, the term “plant part” includes leaves, stems, roots, seed, embryo, pollen, ovules, flowers, root tips, anthers, tissue, cells and the like.
Rebaudioside A (RebA): As used herein “Rebaudioside A” or “RebA” is a steviol glycoside that contains only glucose as its monosaccharide moieties. It contains four glucose molecules in total with the central glucose of the triplet connected to the main steviol structure at its hydroxyl group, and the remaining glucose at its carboxyl group forming an ester bond.
Rebaudioside D (RebD): As used herein, “Rebaudioside D” or “RebD” is an ent-kaurane diterpene glycoside isolated from
Unknown
May 12, 2026
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